Full-length Transcriptome Dataset Research Articles

Desiccation tolerance mechanisms of resurrection plant Selaginella pulvinata

The resurrection plant Selaginella pulvinata possesses an extraordinary ability to survive longterm desiccation, with the capacity to recover to normal growth after four years in a desiccated state. To understand the phenomenon, we investigated morphology, cytology, physiology, and performed PacBio single-molecule, real-time (SMRT) sequencing and Illumina RNA-sequencing of S. pulvinata during dehydration and rehydration. The full-length transcriptome dataset of S. puvinata consisted of 78,914 isoforms, and 75,819 isoforms (96.07%) were functionally annotated. The mapping rate of RNA-seq was 86.83–93.46% when utilizing the full-length transcriptome dataset as a reference. A total of 17,934 differentially expressed genes (DEGs) were identified during dehydration and rehydration. During dehydration, swollen chloroplasts and mitochondria, as well as vacuolation occupied space in desiccated cells, which cooperated with dense cytoplasm to inhibit the collapse and folding of desiccated cells. Accumulation and up-regulated gene expressions of osmotic agent and protective proteins contributed to the formation of vacuolation and dense cytoplasm. To reduce ROS damage during dehydration, two strategies were employed in S. pulvinata, i.e., reducing ROS production and scavenging ROS. Reducing exposure area, inactive PSII and down-regulated gene expression in photosynthesis decreased ROS production caused by excessive light absorption. Increased activities and up-regulated gene expressions in antioxidant enzyme system promoted ROS scavenging. ABA signaling pathway was hypothesized to play an important regulatory role in the desiccation tolerance of S. pulvinata due to significant ABA accumulation and up-regulated expressions of related genes during dehydration. Moreover, the comparative transcriptome analysis revealed that the pathways involved in dehydration and rehydration of the resurrection species S. pulvinata, S. lepidophylla and S. sellowii were consistent, indicating the similar desiccation tolerance mechanisms existing in the different resurrection species of Selaginella. Our study preliminarily revealed the desiccation tolerance mechanisms of S. pulvinata, which holds significant potential for inspiring biotechnological strategies aimed at enhancing drought tolerance in crops.

Iso-Seq combined with RNA-Seq analysis provided insight into the molecular response to dietary oxidized fish oil in juvenile Amur sturgeon, Acipenser schrenckii

Despite the economic and biological importance of Amur sturgeon in the world, limited full-length transcripts are available. Oxidized fish oil (OFO) might induce injury stress, but few related studies were conducted in Amur sturgeon. In this study, an 8-week experiment with diets containing 6% fresh fish oil (FFO) or 6% OFO was conducted, and Iso-Seq combined with RNA-Seq analysis were used to explore the molecular response to dietary OFO in juvenile Amur sturgeon. A total of 18,408 high quality, unique full-length isoforms were generated by Iso-Seq, with an average length of 2180 bp and an N50 of 2793 bp. ~382 million clean reads were obtained from RNA-Seq, and the mapping ratio of the clean reads to the unigenes was 84.25% ~ 88.25%. There were 839 differently expressed genes (DEGs) in FFO vs OFO, of which 360 were up-regulated, and 479 were down-regulated. DEGs were mapped into 276 KEGG pathways, of which 45 pathways were significantly influenced. The proportion analysis of 45 significantly changed pathways revealed that human disease was the most enriched category, and cancers, endocrine system, signal transduction, cell growth and death and carbohydrate metabolism was the most affected subcategory in each category, indicating that OFO might induce cancers, endocrine disorder, and carbohydrate metabolism disruption. Further analysis discovered that transcriptional misregulation in cancer and FOXO signaling pathway were the top2 regulated pathways. PPI network analysis showed that foxo1 and pik3cα were identified as hub genes in response to dietary OFO in Amur sturgeon. The present study provided the first systematic full-length transcriptome dataset of Amur sturgeon and laid a solid foundation for further research on the molecular mechanism in response to dietary OFO.

Unveiling the transcriptomic complexity of Miscanthus sinensis using a combination of PacBio long read- and Illumina short read sequencing platforms

BackgroundMiscanthus sinensis Andersson is a perennial grass that exhibits remarkable lignocellulose characteristics suitable for sustainable bioenergy production. However, knowledge of the genetic resources of this species is relatively limited, which considerably hampers further work on its biology and genetic improvement.ResultsIn this study, through analyzing the transcriptome of mixed samples of leaves and stems using the latest PacBio Iso-Seq sequencing technology combined with Illumina HiSeq, we report the first full-length transcriptome dataset of M. sinensis with a total of 58.21 Gb clean data. An average of 15.75 Gb clean reads of each sample were obtained from the PacBio Iso-Seq system, which doubled the data size (6.68 Gb) obtained from the Illumina HiSeq platform. The integrated analyses of PacBio- and Illumina-based transcriptomic data uncovered 408,801 non-redundant transcripts with an average length of 1,685 bp. Of those, 189,406 transcripts were commonly identified by both methods, 169,149 transcripts with an average length of 619 bp were uniquely identified by Illumina HiSeq, and 51,246 transcripts with an average length of 2,535 bp were uniquely identified by PacBio Iso-Seq. Approximately 96 % of the final combined transcripts were mapped back to the Miscanthus genome, reflecting the high quality and coverage of our sequencing results. When comparing our data with genomes of four species of Andropogoneae, M. sinensis showed the closest relationship with sugarcane with up to 93 % mapping ratios, followed by sorghum with up to 80 % mapping ratios, indicating a high conservation of orthologs in these three genomes. Furthermore, 306,228 transcripts were successfully annotated against public databases including cell wall related genes and transcript factor families, thus providing many new insights into gene functions. The PacBio Iso-Seq data also helped identify 3,898 alternative splicing events and 2,963 annotated AS isoforms within 10 function categories.ConclusionsTaken together, the present study provides a rich data set of full-length transcripts that greatly enriches our understanding of M. sinensis transcriptomic resources, thus facilitating further genetic improvement and molecular studies of the Miscanthus species.

Full-length transcriptome analysis of pecan (Carya illinoinensis) kernels

Pecan is rich in bioactive components such as fatty acids (FAs) and flavonoids and is an important nut type worldwide. Therefore, the molecular mechanisms of phytochemical biosynthesis in pecan are a focus of research. Recently, a draft genome and several transcriptomes have been published. However, the full-length mRNA transcripts remain unclear, and the regulatory mechanisms behind the quality components biosynthesis and accumulation have not been fully investigated. In this study, single-molecule long-read sequencing technology was used to obtain full-length transcripts of pecan kernels. In total, 37,504 isoforms of 16,702 genes were mapped to the reference genome. The numbers of known isoforms, new isoforms, and novel isoforms were 9013 (24.03%), 26,080 (69.54%), and 2411 (6.51%), respectively. Over 80% of the transcripts (30,751, 81.99%) had functional annotations. A total of 15,465 alternative splicing (AS) events and 65,761 alternative polyadenylation events were detected; wherein, the retained intron was the predominant type (5652, 36.55%) of AS. Furthermore, 1894 long noncoding RNAs and 1643 transcription factors were predicted using bioinformatics methods. Finally, the structural genes associated with FA and flavonoid biosynthesis were characterized. A high frequency of AS accuracy (70.31%) was observed in FA synthesis-associated genes. This study provides a full-length transcriptome data set of pecan kernels, which will significantly enhance the understanding of the regulatory basis of phytochemical biosynthesis during pecan kernel maturation.

Identification and expression profiling of genes involved in circadian clock regulation in red dragon fruit (Hylocereus polyrhizus) by full-length transcriptome sequencing

ABSTRACT Crassulacean acid metabolism (CAM) plants fix CO2 at night, exhibiting a reversed regulatory pattern of metabolomic pathways compared with most model plants, which have C3 and C4 pathways. In this study, we used a valuable tropic fruit, red dragon fruit (Hylocereus polyrhizus), as model plant to identify and analyze the circadian regulation genes. Due to the absence of red dragon fruit’s whole-genome dataset, we established a full-length transcriptome dataset using single-molecule real-time (SMRT) sequencing method. A 7.66-Gb dataset with 4,552,474 subreads was generated, with an average length of 1,683 bp and an N50 of 2,446 bp. Using this dataset, we identified center oscillator genes: CCA1 (CIRCADIAN CLOCK ASSOCIATED1), ELF3 (EARLY FLOWERING 3), GI (GIGANTEA), LHY (LATE ELONGATED HYPOCOTYL), LNK1 (NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1), and TOC1 (TIMING OF CAB EXPRESSION 1); a gene for the input pathway: CRY1 (CRYPTOCHROME); a gene for the output pathway: CO (CONSTANS); and genes related to the CAM pathway: MDH (MALATE DEHYDROGENASE), ME (MALIC ENZYMES), and PPDK (PYRUVATE PHOSPHATE DIKINASE). We further established the 24-h rhythmic expression pattern of these genes and classified these into three groups: HpCCA1, HpELF3, HpLHY, HpLNK1, and HpGI have expression peaks during the day; HpTOC1, HpCO, and HpCRY1 have highest expression levels at night; The genes involved in the CAM pathways, namely, HpMDH, HpME1, and HpPPDK, have double expression peaks in the day and night. Comparison of these expression patterns between red dragon fruit and model plants could provide clues in understanding the circadian clock regulation and the activity of the CAM pathways in cactus plants.

Full-Length Transcriptome Analysis of Plasmodium falciparum by Single-Molecule Long-Read Sequencing.

Malaria, an infectious disease caused by Plasmodium parasites, still accounts for amounts of deaths annually in last decades. Despite the significance of Plasmodium falciparum as a model organism of malaria parasites, our understanding of gene expression of this parasite remains largely elusive since lots of progress on its genome and transcriptome are based on assembly with short sequencing reads. Herein, we report the new version of transcriptome dataset containing all full-length transcripts over the whole asexual blood stages by adopting a full-length sequencing approach with optimized experimental conditions of cDNA library preparation. We have identified a total of 393 alternative splicing (AS) events, 3,623 long non-coding RNAs (lncRNAs), 1,555 alternative polyadenylation (APA) events, 57 transcription factors (TF), 1,721 fusion transcripts in P. falciparum. Furthermore, the shotgun proteome was performed to validate the full-length transcriptome of P. falciparum. More importantly, integration of full-length transcriptomic and proteomic data identified 160 novel small proteins in lncRNA regions. Collectively, this full-length transcriptome dataset with high quality and accuracy and the shotgun proteome analyses shed light on the complex gene expression in malaria parasites and provide a valuable resource for related functional and mechanistic researches on P. falciparum genes.

Genetic and Epigenetic Changes Are Rapid Responses of the Genome to the Newly Synthesized Autotetraploid Carassius auratus.

Whole genome duplication events have occurred frequently during the course of vertebrate evolution. To better understand the influence of polyploidization on the fish genome, we herein used the autotetraploid Carassius auratus (4n = 200, RRRR) (4nRR) resulting from the whole genome duplication of Carassius auratus (2n = 100, RR) (RCC) to explore the genomic and epigenetic alterations after polyploidization. We subsequently performed analyses of full-length transcriptome dataset, amplified fragment length polymorphism (AFLP) and methylation sensitive amplification polymorphism (MSAP) on 4nRR and RCC. By matching the results of 4nRR and RCC isoforms with reference genome in full-length transcriptome dataset, 649 and 1,971 novel genes were found in the RCC and 4nRR full-length geneset, respectively. Compared to Carassius auratus and Megalobrama amblycephala, 4nRR presented 3,661 unexpressed genes and 2,743 expressed genes. Furthermore, GO enrichment analysis of expressed genes in 4nRR revealed that they were enriched in meiosis I, whereas KEGG enrichment analysis displayed that they were mainly enriched in proteasome. Using AFLP analysis, we noted that 32.61% of RCC fragments had disappeared, while 32.79% of new bands were uncovered in 4nRR. Concerning DNA methylation, 4nRR exhibited a lower level of global DNA methylation than RCC. Additionally, 60.31% of methylation patterns in 4nRR were altered compared to RCC. These observations indicated that transcriptome alterations, genomic changes and regulation of DNA methylation levels and patterns had occurred in the newly established autotetraploid genomes, suggesting that genetic and epigenetic alterations were influenced by autotetraploidization. In summary, this study provides valuable novel insights into vertebrate genome evolution and generates relevant information for fish breeding.

Full-Length Transcriptome from Camellia oleifera Seed Provides Insight into the Transcript Variants Involved in Oil Biosynthesis

Camellia oleifera Abel., belonging to the genus Camellia of Theaceae, has been widely used as a cooking oil, lubricant, and in cosmetics. Because of complicated polyploidization and large genomes, reference genome information is still lacking. Systematic characterization of gene models based on transcriptome data is a fast and economical approach for C. oleifera. Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) and Illumina RNA-Seq combined with gas chromatography were performed for exploration of oil biosynthesis, accumulation, and comprehensive transcriptome analysis in C. oleifera seeds at five different developmental stages. We report the first full-length transcriptome data set of C. oleifera seeds comprising 40,143 deredundant high-quality isoforms. Among these isoforms, 37,982 were functionally annotated, and 271 (2.43%) belonged to fatty acid metabolism. A total of 8,344 full-length unique transcript models were obtained, and 8,151 (97.69%) of them produced more than two isoforms, suggesting a high degree of transcriptome complexity in C. oleifera seeds. A total of 783 alternative splicing (AS) events were identified, among which the retained intron was the most abundant. We also obtained 1,910 long noncoding RNAs (lncRNAs) and found that AS events occurred in these lncRNAs. Potential transcript variants of genes involved in oil biosynthesis were also investigated. After performing weighted correlation network analysis, we found seven "gene modules" and hub genes for each module showing a significant association with oil content. The series test of clusters classified these modules into four significant profiles based on gene expression patterns. Protein-protein interaction network analysis showed that upregulated WRI1 interacted with 17 genes encoding the enzymes playing key roles in oil synthesis. MYB and ZIP transcriptional factors also showed significant interactions with key genes involved in oil synthesis. Collectively, our data advance the knowledge of RNA isoform diversity in seeds at different developmental stages and provide a rich resource for functional studies on oil synthesis in C. oleifera.

Integrated single-molecule long-read sequencing and Illumina sequencing reveal the resistance mechanism of Psathyrostachys huashanica in response to barley yellow dwarf virus-GAV

Although Psathyrostachys huashanica has excellent potential for resistance gene mining and molecular genetic breeding, no reference genome is available. To date, most studies of P. huashanica have been focused on the creation of translocation lines and additional lines, as well as the development of molecular markers. Therefore, research at the transcriptional level is lacking. In this study, the full-length transcriptome of P. huashanica was sequenced using PacBio isoform sequencing (Iso-Seq) of a pooled RNA sample to explore the potential full-length transcript isoforms. We obtained 112,596 unique transcript isoforms with a total length of 114,957,868 base pairs (bp). Subsequently, Illumina sequencing reads were used to correct and trim the PacBio isoforms. We annotated 103,875 unigenes in at least one functional database, and identified a plethora of differentially-expressed genes (DEGs) that are involved in the defense responses of P. huashanica against barley yellow dwarf virus-GAV (BYDV-GAV). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these DEGs were mostly involved in plant-pathogen interaction, plant hormone signal transduction, and the mitogen-activated protein kinase (MAPK) signaling pathway. Additionally, we selected twenty of the RNA-seq identified resistance-related up-regulated genes, including MAPKs, cysteine-rich receptor-like protein kinases (CRPKs), calcium-dependent protein kinases (CDPKs), pathogenesis-related protein (PR) proteins, WRKYs, and disease resistance proteins, and validated their up-regulation in response to BYDV-GAV by quantitative real-time PCR. Our results indicate that a series of defense-related genes were induced in P. huashanica during BYDV-GAV infection. The full-length transcriptome dataset will contribute to improved use of stress-resistance genes of P. huashanica, and serves as a reference database for the analysis of transcript expression in P. huashanica.

Full-length transcriptome sequences of Agropyron cristatum facilitate the prediction of putative genes for thousand-grain weight in a wheat-A. cristatum translocation line

BackgroundAgropyron cristatum (L.) Gaertn. (2n = 4x = 28; genomes PPPP) is a wild relative of common wheat (Triticum aestivum L.) and provides many desirable genetic resources for wheat improvement. However, there is still a lack of reference genome and transcriptome information for A. cristatum, which severely impedes functional and molecular breeding studies.ResultsSingle-molecule long-read sequencing technology from Pacific Biosciences (PacBio) was used to sequence full-length cDNA from a mixture of leaves, roots, stems and caryopses and constructed the first full-length transcriptome dataset of A. cristatum, which comprised 44,372 transcripts. As expected, the PacBio transcripts were generally longer and more complete than the transcripts assembled via the Illumina sequencing platform in previous studies. By analyzing RNA-Seq data, we identified tissue-enriched transcripts and assessed their GO term enrichment; the results indicated that tissue-enriched transcripts were enriched for particular molecular functions that varied by tissue. We identified 3398 novel and 1352 A. cristatum-specific transcripts compared with the wheat gene model set. To better apply this A. cristatum transcriptome, the A. cristatum transcripts were integrated with the wheat genome as a reference sequence to try to identify candidate A. cristatum transcripts associated with thousand-grain weight in a wheat-A. cristatum translocation line, Pubing 3035.ConclusionsFull-length transcriptome sequences were used in our study. The present study not only provides comprehensive transcriptomic insights and information for A. cristatum but also proposes a new method for exploring the functional genes of wheat relatives under a wheat genetic background. The sequence data have been deposited in the NCBI under BioProject accession number PRJNA534411.

Analysis of Chromosomal Numbers, Mitochondrial Genome, and Full-Length Transcriptome of Onychostoma brevibarba

Onychostoma brevibarba is a new discovered species which is distributed in Xiang Jiang River of the middle Chang Jiang basin in Hunan Province, South China. In this study, the ploidy levels of O. brevibarba were confirmed by counting chromosomal numbers and analyzing karyotype. The complete mitochondrial genome of O. brevibarba was determined and analyzed. Besides, we firstly performed the full-length transcriptome of O. brevibarba derived from 5 different tissues using the PacBio SMRT sequencing. The result shows that O. brevibarba was a diploid with 48 chromosomes. The complete mitogenome of O. brevibarba was 16,602 bp in size and very similar (89.1–91.3%) to that of other Onychostoma species but was distinct from all congeners. The full-length transcriptome dataset of O. brevibarba comprised 120,239 unigenes. Among the unigenes, 91,542 were functionally annotated, whereas 26,794 were found to have two or more isoforms. This study could provide many new insights into cytology and molecular characteristics of O. brevibarba; it laid the foundation for further exploration of the genomic signatures of species of Onychostoma.

Transcriptomic profiles of 33 opium poppy samples in different tissues, growth phases, and cultivars

Opium poppy is one of the most important medicinal plants and remains the only commercial resource of morphinan-based painkillers. However, little is known about the regulatory mechanisms involved in benzylisoquinoline alkaloids (BIAs) biosynthesis in opium poppy. Herein, the full-length transcriptome dataset of opium poppy was constructed for the first time in accompanied with the 33 samples of Illumina transcriptome data from different tissues, growth phases and cultivars. The long-read sequencing produced 902,140 raw reads with 55,114 high-quality transcripts, and short-read sequencing produced 1,923,679,864 clean reads with an average Q30 rate of 93%. The high-quality transcripts were subsequently quantified using the short reads, and the expression of each unigene among different samples was calculated as reads per kilobase per million mapped reads (RPKM). These data provide a foundation for opium poppy transcriptomic analysis, which may aid in capturing splice variants and some non-coding RNAs involved in the regulation of BIAs biosynthesis. It can also be used for genome assembly and annotation which will favor in new transcript identification.

Full-length RNA sequencing reveals unique transcriptome composition in bermudagrass

Bermudagrass [Cynodon dactylon (L.) Pers.] is an important perennial warm-season turfgrass species with great economic value. However, the reference genome and transcriptome information are still deficient in bermudagrass, which severely impedes functional and molecular breeding studies. In this study, through analyzing a mixture sample of leaves, stolons, shoots, roots and flowers with single-molecule long-read sequencing technology from Pacific Biosciences (PacBio), we reported the first full-length transcriptome dataset of bermudagrass (C. dactylon cultivar Yangjiang) comprising 78,192 unigenes. Among the unigenes, 66,409 were functionally annotated, whereas 27,946 were found to have two or more isoforms. The annotated full-length unigenes provided many new insights into gene sequence characteristics and systematic phylogeny of bermudagrass. By comparison with transcriptome dataset in nine grass species, KEGG pathway analyses further revealed that C4 photosynthesis-related genes, notably the phosphoenolpyruvate carboxylase and pyruvate, phosphate dikinase genes, are specifically enriched in bermudagrass. These results not only explained the possible reason why bermudagrass flourishes in warm areas but also provided a solid basis for future studies in this important turfgrass species.